15

CHAPTER 8-3 NUTRIENT RELATIONS: NITROGEN

Figure 1. Physcomitrella patens growing on previously flooded soil. Note the nitrogen-fixing blue-green bacterium, Nostoc, at the arrow. Photo by Michael Lth.

N Forms
Nitrogen is available in many forms. The most abundant of these, N2 gas, cannot be used by plants or animals and must be converted by Cyanobacteria or bacteria before plants can use it. Animals can only obtain it by eating other organisms that have already placed the N into amino acids. Other forms of N that plants can absorb include ammonium (NH4+), nitrite (NO2-), nitrate (NO3), and organic forms such as amino acids and urea. As we shall soon see, not all plants have the same ability to use these forms and some are toxic to most taxa. Nitrate and Ammonium Plants, including bryophytes, can take in and use both NO3- (nitrate) and NH4+ (ammonium). The form of nitrogen needed by bryophytes varies with species and habitat. Aquatic higher plants use nitrogen in three inorganic forms: NO2- (nitrite) (Schwoerbel & Tillmanns 1964, 1977), NO3-, NH4+ (Schwoerbel & Tillmanns 1972; Rudolph & Voigt 1986). Bryophytes usually absorb NH4+ more easily than they absorb NO3- (Schwoerbel & Tillmanns 1974; Simola 1975; Miyazaki & Satake 1985; Schuurkes et al. 1986). Vanderpoorten (2000) reported that NH4+ N is one of the best factors to explain differences in aquatic Amblystegium distributions in river systems. Frahm (1975) found that the brook moss Fontinalis antipyretica var. gigantea had a low tolerance for NH4+, but Schwoerbel and Tillmanns (1974, 1977) found conflicting evidence showing that this species uses NO3and NH4+, with NH4+ being taken up first if provided together with NO3-. In fact, it is unable to uptake NO3- in the dark (Schwoerbel & Tillmanns 1974). It is possible that various strains have developed within species that have different tolerance levels for some of their nutrients. The report of amino acid utilization in the aquatic Java moss (Taxiphyllum barbieri; Alghamdi 2003) seems unusual among the aquatic mosses and may somehow relate to its ability to live in aquaria and tropical streams where most

16

Chapter 8-3: Nutrient Relations: Nitrogen

other bryophytes seem unable to survive. Could this in some way relate to the higher annual temperatures of its tropical habitat? Assuming that bryophytes operate as do tracheophytes, NO3-, once in the plant, is converted to NH4+. In leaves, the intermediate product, NO2-, is reduced by nitrite reductase (an enzyme that facilitates the addition of hydrogen and loss of oxygen from NO2- during the photosynthetic electron transport process). No intermediate product is released and the final product is NH4+. Since photosynthesis provides the NADH (nicotinamide adenine dinucleotide, the active coenzyme form of vitamin B3) and ferredoxin needed for conversion of nitrogen oxides to NH4+, the conversion process is enhanced by the same things that enhance photosynthesis high light and warm temperatures (Salisbury & Ross 1978). Thus, more ammonium is produced. NO3nitrate

This relationship was consistent with much greater growth at 30 mg/L N than at 10 mg/L N as NH4+ (Figure 2).

NO2nitrite

nitrite reductase

NH4+
ammonium

Brown (1982) suggested that the pH or alkalinity affects availability of N for plants, with NO3- being more available in neutral or alkaline soils and NH4+ in acidic soils and water. But NH4+ is usually toxic to plants in any appreciable quantity. Sironval (1947) found that NH4+ ions caused degeneration of the caulonema of Funaria hygrometrica and Southorn (1977) found they caused morphological abnormalities in the same species. Killian (1923) likewise found morphological abnormalities in the leafy liverwort Scapania. On the other hand, Burkholder (1959) found that cultured bryophytes did equally well on both NO3- and NH4+ salts. An interesting consequence of pH differences was suggested by Machlis (1962). In Sphaerocarpos texanus, male plants are smaller than females in the field. Machlis attributed this to the ability of male plants to absorb NH4+ ions more readily than females, causing them to have a lower pH, which could suppress growth. He supported this suggestion by growing the plants on potassium, which caused no pH change, and likewise no reduction in the size of male plants. In a study designed to determine the effects of various forms of N on bryophyte function, Alghamdi (2003) studied the popular, fast-growing aquarium moss Taxiphyllum barbieri (Java moss). He found that the benefit to the moss depends on what parameter you measure (Figure 2). For example, dry biomass increase was greatest in high NO3- concentrations (30 mg/L N), whereas the greatest increase in length occurred in high NH4+ concentrations (30 mg/L N). This difference resulted in the least biomass increase per stem length in high NH4+ concentrations, despite the relatively high increase in length in that treatment. The overall appearance of the mosses in high NH4+, then, was to appear long and thin compared to those in other treatments, but not dissimilar to the plants in the control (standard nutrient solution but with no N source). Based on the lower growth in the NH4NO3 media, Alghamdi reasoned that in the presence of NH4+, the NO3- became unusable because of the inhibition of nitrate reductase by NH4+. At the same time, the lower concentration of NH4+ (15 mg/L N) in combination compared to NH4+ alone (30 mg/L N) reduced the growth.

Figure 2. Effects of various forms of inorganic N (control = no N, NO2- = nitrite, NO3- = nitrate, NH4+ = ammonium) on growth in length and biomass of Taxiphyllum barbieri. Box mean (dot) and median (horizontal line); bottom of box is first quartile and top is third quartile. Whiskers represent lowest and highest observations still inside region defined by lower limit Q1 - 1.5 (Q3 - Q1) and upper limit Q3 + 1.5 (Q3 - Q1); * represents outliers that extend beyond the whiskers; n = 15 sets of 3 stems. Means with same letters are not significantly different from each other (DNMRT, = 0.05). Based on Alghamdi (2003).

NO2- caused only modest improvements in biomass and length over the N-free controls (Figure 2), but caused considerable increase in chlorophyll a (Alghamdi 2003; Figure 3). The chlorophyll a:b ratio was highest in the high NO3- treatment, due to mosses in that treatment having the least chlorophyll b per biomass of moss, a concentration even lower than that of the controls (Figure 3). In fact, the effects of inorganic N form on chlorophyll b resulted in either no improvement over N-free controls, or depressed levels of chlorophyll b. Chlorophyll a, on the other hand, was higher in nearly all the nitrogen treatments than in the controls. Baxter et al. (1992) found a similar but slight decrease in total chlorophyll concentration in Sphagnum cuspidatum, typically a submersed species, with increasing levels of NH4+, but in Alghamdi's experiments,

Chapter 8-3: Nutrient Relations: Nitrogen

17

Taxiphyllum barbieri actually had a total chlorophyll increase, although not statistically significant, with an increase from 1 to 30 mg/L N as NH4+ (Figure 3). Protein concentrations in Taxiphyllum barbieri showed a very different picture from other measurements, with little difference among treatments except at 10 and 30 mg/L NO3- (Figure 3; Alghamdi 2003). In Sphagnum cuspidatum the addition of NH4+ (as NH4Cl) generally caused an increase in amino acids, at least within the first 15 days, in both locations studied, with arginine increasing the most. at the unpolluted site and actually decreasing at the NH4+polluted site (Baxter et al. 1992). The latter study suggests that Sphagnum cuspidatum may acclimate to a higher level of NH4+ in a way that it eventually requires higher levels than populations not continuously exposed to such high levels. Clearly the uses of the various forms of N in bryophytes are complex and one cannot give a simple answer as to which form is best.

Figure 3. Effects of various forms of inorganic N (control = no N, NO2- = nitrite, NO3- = nitrate, NH4+ = ammonium) on chlorophyll a and protein concentrations of Taxiphyllum barbieri. Notation as in Figure 2; n = 15 sets of 3 stems. Based on Alghamdi (2003).

In Sphagnum, differences exist among the species. Sphagnum flexuosum is apparently unable to utilize NO3(Schuurkes et al. 1986), and Touffet (1971) found that NO3- actually reduced the growth of Sphagnum and was less effectively utilized than NH4+ when it was the only N resource. Nevertheless, in many Sphagnum species nitrate reductase, an inducible enzyme (Deising 1987), permits use of NO3-. High levels of NH4+ inhibit nitrate reductase, and hence reduce growth, by inhibiting NO3- uptake (Rudolph et al. 1987). Rudolph and Voigt (1986) demonstrated that 322 M was a favorable concentration of NO3- in Sphagnum magellanicum, whereas at 225 M NH4+ the chlorophyll content decreased. At 600 M NH4+, the nitrate reductase activity was reduced up to 20%. These factors most likely contribute to the location of mosses in their particular habitats by defining their nutrient niches.

Organic Nitrogen Most agricultural plants seem to absorb their nitrogen in the form of NH4+ or NO3-, but it seems that bryophytes have more options. Sphagnum is able to use urea (along with phosphate) in the Alaskan wetlands, resulting in an increase in biomass compared to controls (Sanville 1988). In nature, amino acids likewise can be abundant, present as breakdown products of plant and animal wastes and litter. Yet few culture studies or field tracer studies have included these organic forms. Is it possible that bryophytes can use this organic N as their primary source? If so, they may benefit from organic leachates in early stages of litter decomposition of a soil environment. In bogs and poor fens, NH4+ seems to be the predominant form of available N (Rosswall & Granhall 1980). NO3- is often lost through denitrification (Hemond 1983). Not surprisingly, some studies show that Sphagnum seems to require most of its inorganic N as NH4+ (Schuurkes et al. 1986). But Simola (1975, 1979) showed that Sphagnum nemoreum and S. fimbriatum both could use amino acids. Sphagnum nemoreum grows almost as well on the amino acids arginine and alanine as on NH4+ salts, but it is unable to use leucine, lysine, or methionine (Simola 1975). More recently, McKane (1993), using tracer studies, found that for Sphagnum, Aulacomnium palustre, and Hylocomium splendens, the amino acid glycine was actually the preferred form of nitrogen over NH4+ and NO3-. It appears that in Arctic ecosystems, organic nitrogen (amino acids, especially glycine) may actually be the preferred source of N for some bryophytes, including Sphagnum rubellum (Kielland 1997). Even amino acids with higher molecular weights, such as aspartate and glutamate, can be absorbed at higher rates than inorganic N. Kielland suggested that the high capacity for absorbing amino acids might be an adaptation to the low inorganic N availability in the Arctic. Even floodplain bryophytes can use amino acids. Schuler et al. (1955) found that in culture the thallose liverwort Sphaerocarpos texanus grew more typically on a mix of amino acids than it did on NH4NO3 alone. Brown (1982) suggested that in low N environments the mosses may be able to move organic molecules containing N from dying and dead cells to the growing apex. It is very likely that these molecules would be amino acids, as well as dipeptides and other organic compounds. Burkholder (1959) examined the effects of 20 amino acids (0.0001 M AA to 0.0016 M AA with and without the addition of NH4NO3 on the color and growth of Atrichum undulatum. Glycine, L-cystine, L-cysteine, and Ltyrosine were the only treatments with amino acids alone in which the moss retained its green color. Others were yellow-green, brown-green, or brown (DL-serine and DLtryptophan). When grown in combination of each of these 20 amino acids with NH4NO3, plants in all treatments grew more than in any of the amino acids alone except in the highest concentration (0.0016 M) of DL-tryptophan. Growth was generally greatest in the lower concentration of amino acid (0.0001 M) plus NH4NO3. Alghamdi (2003) chose common soil water-soluble amino acids (glycine, methionine, serine, arginine, and alanine) to compare their effects on growth, branching, chlorophyll, and protein on Taxiphyllum barbieri. He

18

Chapter 8-3: Nutrient Relations: Nitrogen

found that four of these amino acids induced branching, relative to the controls, but no branching appeared in any of the methionine treatments (Figure 4).

dry biomass to length, but maintained a length somewhat less than that of the N-free controls (Figure 5). This resulted in unusually short, wide plants, combined with high protein concentrations but below normal chlorophyll concentrations at the lowest level applied (1 mg/L; Figure 6). Methionine likewise caused an increase in biomass and decrease in length growth with concentration increase (1, 10, 30 mg/L). Alanine caused an increase in both length and biomass with concentration, with the overall effect being one of a more robust plant at higher concentrations, having a higher biomass to length ratio than that of the controls. The mosses responded to 1 mg/L glycine much as they did to the N-free medium, but at higher concentrations (20 and 30 mg/L) their length and biomass both increased considerably over that of controls.

Figure 4. Effects of water soluble amino acids on number of branches in the Java moss, Taxiphyllum barbieri. cont = control, gly = glycine, meth = methionine, ser = serine, arg = arginine, ala = alanine. From Alghamdi (2003).

Figure 6. Effect of water soluble amino acids on the protein content and total chlorophyll concentration of the Java moss, Taxiphyllum barbieri. cont = control, gly = glycine, meth = methionine, ser = serine, arg = arginine, ala = alanine. n = 10 sets of 3 stems. Notation as in Figure 2. From Alghamdi (2003).

Figure 5. Effect of water soluble amino acids on the biomass, length, and robustness (wt:length) of the Java moss, Taxiphyllum barbieri. Cont = control, Gly = glycine, Meth = methionine, Ser = serine, Arg = arginine, Ala = alanine. Length and biomass represent sum of 3 stems; n = 10 sets of 3 stems. Notation as in Figure 2. Based on Alghamdi (2003).

Methionine proved to be inhibitory to growth in length whereas serine caused an increase in both dry biomass and length relative to controls (Figure 5; Alghamdi 2003). Arginine as the only N source at 1, 10, and 30 mg/L caused a striking increase in the biomass and ratio of

Alghamdi (2003) then compared the effects of glycine, which seemed to produce the "healthiest" plants, to those of the inorganic forms of N. This aquatic moss did less well on the inorganic forms NH4NO3 or NO3- than on NH4+ alone or NH4+ + the amino acid glycine and did best on glycine alone, producing more biomass, longer stems, and more branches (Figure 7, Figure 8). In fact, glycine seemed to induce branching (Table 1). In the same series of experiments, Alghamdi (2003) examined the effects of inorganic N and glycine on the chlorophyll and protein content. Glycine, both alone and in combination with NH4+, resulted in the highest protein concentrations (Figure 9). The effects on chlorophyll were less clear, but the highest total chlorophyll occurred in the highest glycine concentration. NH4+ at 20 mg/L, however, produced similar chlorophyll concentrations, but at 30 mg/L the chlorophyll content decreased.

Chapter 8-3: Nutrient Relations: Nitrogen

19

Figure 7. Effect of N source as nitrate (NO3+), ammonium (NH4+), glycine (gly), and combinations at two concentrations on number of branches in Taxiphyllum barbieri. The combinations have half the total N from each source. From Alghamdi (2003).

growth in a medium with yeast nucleic acids as its N source, Burkholder (1959) tested growth of this species on the nucleic acid bases. Growth of leafy shoots was good in adenine and guanine, but there was no growth in uracil or thymine. Growth in xanthine, uric acid, and cytosine was less than that in NH4NO3. Both uracil (in the presence of NH4NO3) and aspartic acid caused Sphagnum squarrosum to become thalloid (resembling its protonema), as did hydroxyproline + glycine, occasionally (Burkholder 1959). Not all mosses responded in the same way. Growth of Leptobryum pyriforme and Splachnum sphaericum and others was "excellent" on a medium with NH4NO3 plus uracil, but was poor in Sphagnum squarrosum. On the other hand, while growth of Leptobryum pyriforme was good with uric acid and cytosine, Splachnum sphaericum had poor growth. The ability to use nucleic acids, amino acids, and other organic N compounds could permit bryophytes to take advantage of partially decomposed litter in which these nitrogen sources leak from the dead tissues.
Table 1. Effect of various N forms on moss branching in Taxiphyllum barbieri. From Alghamdi (2003).

Treatment glycine NO3NH4+ glycine + NH4+ NH4NO3

Moss Branching long with many short branches short and no branches long and few short branches long with many short branches and slightly thin short, thin and few short branches

Figure 8. Effect of nitrate (NO3+), ammonium (NH4+), glycine (gly), and combinations on the increase in biomass and length and robustness (wt:length) of the Java moss, Taxiphyllum barbieri. Notation as in Figure 2; n = 10 sets of 3 stems. From Alghamdi (2003).

Other organic compounds, such as nucleic acids, are also released from organism tissues as they decay. Based his data showing that Atrichum undulatum had good

Figure 9. Effects of inorganic N compared to glycine on the protein and chlorophyll content of Java moss (Taxiphyllum barbieri). Notation as in Figure 2; n = 10 sets of 3 stems. From Alghamdi (2003).

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Chapter 8-3: Nutrient Relations: Nitrogen

Amino acids, leaking into the environment, could cause developmental anomalies leading to abnormal growth forms in bryophytes. Some amino acids, such as hydroxyproline, can cause desuppression in the development of underleaves in liverworts (Basile & Basile 1980; Basile et al. 1988), causing them to look like normal leaves. In Atrichum, amino acids inhibited leafy shoot development (Burkholder 1959). This might be another example of the Gaia hypothesis (Lovelock, 1988), wherein the ecosystem behaves like a superorganism and species depend on other species for their biochemical needs during development.

Figure 11. Nitrogen fixation in Cyanobacteria, with atmospheric nitrogen entering an adjacent cell and being transferred to the heterocyst, where it is converted to ammonium (NH4+). The ammonium is then moved to the adjacent cell where it is converted into organic compounds, typically amino acids. Diagram by Janice Glime.

Nitrogen Fixation
With 78% of our atmosphere being composed of nitrogen and only about 5% of biomass being nitrogen, one would expect this element to be no problem for living systems to obtain. But unlike phosphorus, it cannot normally be obtained from bedrock. And just as you and I can make no use of the free, gaseous nitrogen we breathe, most plants can't either. Instead, plants require their nitrogen fixed into ammonium (NH4+) or nitrate (NO3-) salts (or converted to amino acids) before they can obtain and convert it to specific amino acids and proteins they need. Nitrogen fixation is the process of trapping atmospheric nitrogen and converting it to NH4+ and in some cases, converting it to NO3-. Nitrogen fixation is a major source of usable nitrogen, particularly in bogs and fens. Like many tracheophytes, bryophytes can use N released by N fixation from associated bacteria and Cyanobacteria. The heterocysts (Figure 10) of Cyanobacteria make them a rich source of amino acids as a result of their nitrogen-fixing activity. That is, they are able to convert atmospheric N to a form usable by other living organisms.

Many studies have shown that some bryophytes, especially peatland bryophytes, obtain N through N fixation processes of surface-dwelling Cyanobacteria as well as other bacteria (Cullimore & McCann 1972; Granhall & Selander 1973; Alexander et al. 1974; Basilier et al. 1978; Smith & Ashton 1981; Smith 1984; Nakatsubo & Ino 1986, 1987; Bentley 1987; Given 1987; Bergman et al. 1993; Madhusoodanan & Dominic 1996). In the Cyanobacteria, the most significant contributions come from taxa such as Nostoc (Figure 1), Anabaena, and Calothrix (Figure 12) that have special cells called heterocysts. These cells provide a "safe" environment for nitrogen fixation because they lack the oxygen-generating reactions of photosystem II. The enzyme nitrogen reductase is unable to make the conversion in an aerobic environment, hence requiring a location where photosynthetic oxygen is not available. Since only the Cyanobacteria and some true bacteria are able to use the abundant atmospheric nitrogen, this conversion makes a significant contribution to usable nitrogen in the ecosystem.

Figure 10. Anabaena (Cyanobacteria) showing heterocyst in middle lower part of picture. Photo by Janice Glime.

In the process of nitrogen fixation in Cyanobacteria, the simple CH2O group from sugars, fixed by cells adjacent to the heterocyst, is moved into the heterocyst (Figure 11). Atmospheric nitrogen (N2) enters adjacent cells and is passed to the heterocyst. In the heterocyst nitrogen reductase (enzyme that catalyzes addition of H+ to N to form NH4+) catalyzes the transformation of N2 to the reduced NH4+ with H+ obtained from the CH2O group.

Figure 12. Upper: Algae on Campylopus at geothermal vent in New Zealand. Lower: Nostoc, a typical N-fixing Cyanobacterium that can be found associated with bryophytes. Photos by Janice Glime

Chapter 8-3: Nutrient Relations: Nitrogen

21

These Cyanobacteria fix more nitrogen than is essential for their own needs and release the excess to their environment. Significant contributions of N through N fixation by Cyanobacteria occur in grasslands (Vlassak et al. 1973), boulder communities (Snyder & Wullstein 1973a, Jones & Wilson 1978), tropical forests, especially in epiphyllous communities (those growing on a leaf) (Bentley 1987), poor Sphagnum mires (Basilier 1979), and polar turfs (Alexander et al. 1978). In the terrestrial moss Hymenostylium recurvirostre, association with Nostoc is common. Labelled 15N from N gas, converted by Nostoc, resulted in the highest concentrations in the new rhizoids, then new shoots, then old shoots and old rhizoids (Jones & Wilson 1978). Jones and Wilson suggest that these locations indicate the nitrogen is being translocated from old to young tissues. Not only is free NH4+ available, but also large quantities of extracellular amino acid leakage is associated with this Nostoc. In view of the discussion above on bryophyte use of amino acids, it is likely that the moss and its neighbors might be using these amino acids as part of their N source. In some of the liverworts and hornworts, Cyanobacteria seem to behave symbiotically (Saxena 1981), but more frequently it seems to be only a matter of suitable habitat. For example, in the moist Pacific northwest, approximately 85% of the sampled epiphytic leafy liverwort Porella navicularis (Figure 13) harbors Nostoc (Cyanobacteria) in distinct colonies under the leaf margins and in other plant crevices (Dalton & Chatfield 1985). Nitrogen fixation is measured by the acetylene reduction method, and the product C2H2 is used as the measure of fixation. The production of fixed N on P. navicularis resulted in a mean of 53.5 nmol C2H2 g-1 d m h1 and reached up to 316 nmol C2H2 g-1 d m h-1. Dalton and Chatfield (1985) at first thought the Porella association was symbiotic, but the low number of heterocysts (3-7%) is typical of free-living Nostoc; symbiotic ones typically have a frequency of 30-40%. In either case, the effect is the same; by providing a suitable habitat for Cyanobacteria, the mosses facilitate an increase of available N in the system. Nevertheless, at least some micro-organisms living in association with epiphyllous liverworts are able to transfer this fixed nitrogen directly to their host plants (Figure 14; Bentley & Carpenter 1984), thus constituting a loose arrangement that benefits the tracheophyte as well as the bryophyte. In the palm Welfia georgii, 10-25% of the N in the leaf was derived from the micro-organisms harbored there among the leafy liverwort cover. In bryophyte-Cyanobacteria associations in the Antarctic (Smith & Russell 1982; Smith 1984; Nakatsubo & Ino 1987, Line 1992; Pandey et al. 1992), Arctic (Alexander et al. 1978) and alpine/subalpine zones (Lambert & Reiners 1979), N fixation may be a very important contribution of this limiting nutrient to the ecosystem (Smith & Ashton 1981). Although Smith and Ashton failed to show much acetylene reduction to indicate fixation activity in the field at ~0C, they considered that during the warm summer, fixation by Cyanobacterial flora of bryophytes could approach that exhibited in the lab at ~20C, thus contributing significantly to the available N in the ecosystem. In a 48-hour field incubation with an air temperature of 1.7C and moss moisture of 300-1500%, only the moss Ditrichum strictum associations had any

positive acetylene reduction (1.17 & 1.21 g g-1 48h-1). The more protected, but nevertheless very cold, Clasmatocolea humilis and Jamesoniella grandiflora associations failed to demonstrate any fixation.

Figure 13. Porella navicularis growing epiphytically on a branch. Photo by Kent Brothers with permission from www.botany.ubc.ca/ bryophyte/LAB8.htm.

Figure 14. Means and standard errors of 5 hrs of production of fixed nitrogen in leaves of the palm Welfia georgii incubated alone (with epiphylls removed) and leaves with intact epiphylls, indicating a much greater transfer of new N to the leaf when epiphylls are present. Redrawn from Bentley & Carpenter (1984).

In support of the suggestion that contributions in the summer may be significant, Nakatsubo and Ino (1987) found that approximately 330 mg N m-2 was fixed per growing season in some areas of the Antarctic. Fogg and

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Chapter 8-3: Nutrient Relations: Nitrogen

Stewart (1968) found that most N fixation occurs at temperatures above 10C, thus explaining the lack of activity in the Smith and Ashton study. Temperatures in the moss-Cyanobacterial associations in summer in the maritime Antarctic typically are in excess of 10C, often reaching 20C during midday (Huntley 1971). Smith (1984) found that the fixation rate increased at temperatures from 5C to a maximum at 25-27C, decreasing sharply after that. Saturation occurred at ~1000 mol m-2s-1 photon flux density, decreasing at higher levels. Once suitable temperatures were available, moisture seemed to be the most important criterion, causing an increase in fixation up to the highest water content measured: 3,405%! The chemical conditions suitable for fixation seem to be restrictive, with an optimum pH in this system of 5.9-6.2 and a negative response to the addition of P, Co, or Mo (Smith 1984). Hence, under warmer conditions, fourteen out of nineteen bryophyte associations did indeed exhibit fixation, with values increasing as moisture content increased (Smith & Russell 1982). Rates ranged from 0.36 to 310.57 nmol C2H2 g-1 d m h-1 among the fourteen with measurable fixation. In the Arctic soils of Svalbard, Norway, N fixation by both free-living and bryophyte associations of Cyanobacteria is the only significant source of N input to the soil ecosystem (Solheim et al. 1996). The most important bryophytes for harboring such associations were Calliergon richardsonii and Sanionia uncinata. An interesting factor in the fixation was grazing by geese. Grazed areas had a 10-fold maximum fixation (693.6 1.5 nmol C2H4 h-1 gdm-1) compared to ungrazed areas (65.3 16.6 nmol C2H4 h-1 gdm-1), perhaps because in these areas the Cyanobacteria also occurred on the grass. The transfer of fixed N to the plants supported high plant productivity. On the other hand, where birds harbored under cliffs, the concentration of bird droppings inhibited N fixation. The alpine zone likewise is nitrogen limited due to the slow decay rate and limited organic layer. Cyanobacteria are important in binding the soil and in providing reduced N. In the subalpine zone of the White Mountains of New Hampshire, USA, the moss Plagiomnium cuspidatum provides a suitable habitat for Cyanobacteria (Lambert & Reiners 1979). On Mt. Fuji, the moss communities of the dry SW slope are nearly devoid of N-fixing activity, but on the moist NE-facing cliffs they exhibit high activity, especially with Nostoc colonies (Nakatsubo & Ohtani 1991), again demonstrating the importance of moisture. In the somewhat less severe climate of the Alaskan blue spruce taiga system, feather mosses such as Pleurozium schreberi and Hylocomium splendens are important substrates for N-fixing aerobic and facultative anaerobic bacteria (Billington & Alexander 1983). But the importance of the Cyanobacteria and other Nfixing micro-organisms is not limited to such cold environments. N fixation by Cyanobacterial associations with bryophytes may be important in many ecosystems where it has hardly been recognized (Cullimore & McCann 1972; Madhusoodanan & Dominic 1996). In rainforests, epiphyllous liverworts provide the moist microhabitat needed for high rates of nitrogen fixation by associated bacteria and Cyanobacteria (Bentley & Carpenter 1980; Bentley 1987; Carpenter 1992), which may be transferred

to the host leaves (Bentley & Carpenter 1984). In cryptogamic crusts (i.e. soil crusts of algae, lichens, bryophytes, and micro-organisms; Figure 15) of prairies, deserts, and grasslands, Cyanobacteria are able to maintain an active state longer when water is held by the bryophytes. This increases their contribution to the usable N in the soil (Vlassak et al. 1973; Giddens 1982; Belknap et al. 2001). The crust itself is vital to maintaining both water and nutrients in the soil during and following heavy storms. In geothermal fields and following fires, bryophytes again provide the moist environment needed to maintain N-fixing micro-organisms (Brasell et al. 1986). Hence, we must ask if the bryophytes are net users of nitrogen, or do they facilitate a net gain to the system. At least in some habitats they definitely facilitate a gain by providing the right habitat for fixation to occur.

Figure 15. Cryptogamic crust with the moss Syntrichia inermis. Photo by Lloyd Stark.

Although moss associates are responsible for most N fixation in Arctic and subarctic ecosystems, legume associations are considered the predominant N fixers in temperate ecosystems (Stewart 1967). Nevertheless, in some temperate habitats bryophytes are the only plants able to occupy the habitat. For example, on granite outcrops, bryophytes, especially Grimmia/Schistidium, are well known for their role in accumulating soil and nutrients and holding the moisture needed for tracheophyte establishment. Microbial nitrogen fixation on these bryophytes is part of this successional story (Snyder & Wullstein 1973a; Jones & Wilson 1978). Likewise, bryophyte-Cyanobacteria associations are important in the colonization of volcanic lava. Cyanobacteria are common on bryophytes of dry lava fields (Englund 1976) as well as on the moist, warm bryophyte surfaces near steam vents (Broady et al 1987). Both Anabaena variabilis and Nostoc muscorum were associated with Funaria hygrometrica on the newly formed volcano Surtsey off the Icelandic coast (Rodgers & Henriksson 1976). Although the Funaria did not directly affect the fixation rate, growth of both the Funaria and the Cyanobacteria benefitted by the association, and the N content of Funaria also increased as a result of the cyanobacterial N fixation. Thus, as in the Arctic, temperate bryophytes often have associated Cyanobacteria, especially Nostoc. Soil associations with bryophytes can benefit the ecosystem in several ways. Not only do they provide additional usable N to the ecosystem, as in the Gymnostomum recurvirostrum

Chapter 8-3: Nutrient Relations: Nitrogen

23

association in Upper Teesdale (Wilson 1975), but they also provide a buffer against erosion and leaching of nutrients already in the upper soil layers. Few studies have quantitatively addressed the role of micro-organisms in bryophyte communities, particularly in

peatlands where their role is significant (Gilbert et al. 1999). Nevertheless, these micro-organisms are undoubtedly key players in nutrient cycling through the microbial loop.

Table 2. Comparison of N fixation rates by Cyanobacteria associated with bryophytes in various habitats. Rates converted to nmol N using the 3:1 ratio of reduced acetylene to fixed N given by Nakatsubo and Ino (1987) and Vlassak et al. (1973). gfm = grams fresh mass; gdm = grams dry mass. Table compiled by Medora Burke-Scoll.

Location
Tropical Tropical Tropical Tropical Tropical Tropical Tropical Tropical Temperate Temperate Japan Temperate Temperate Temperate Temperate Temperate Temperate Temperate Temperate Temperate Boreal Iceland Boreal Subalpine Subalpine Subalpine Subalpine Subalpine Subalpine Subalpine Subalpine Subalpine Subalpine

Habitat
Lava and on volcanic island Undisturbed forest floor Undisturbed forest floor Undisturbed forest floor Undisturbed forest floor Undisturbed forest floor Undisturbed forest floor Undisturbed forest floor Grassland Aquatic Peatland Coniferous forest floor (Bilberry-spruce forest) Forest margin Fen Fen Fen Lakeside Desert Desert Iceland Lava field Iceland Lava field Forest floor Peatland Peatland Aquatic Forest floor Forest floor Forest floor Forest floor Forest floor Forest floor

Bryophyte and Cyanobacteria partner

Funaria hygrometrica + Nostoc & Anabaena Chiloscyphus coalitus + Anabaena &/or Nostoc Chiloscyphus fissistipus + Anabaena &/or Nostoc Bazzania adnexa + Anabaena &/or Nostoc Hypnum chrysogaster + Anabaena &/or Nostoc Pohlia nutans + Anabaena &/or Nostoc Tortella calycina + Anabaena &/or Nostoc Pohlia nutans + Anabaena &/or Nostoc Ceratodon purpureus + Nostoc Sphagnum capillaceum + Stigonema, Hapalosiphon, Scytonema, & Nodularia Sphagnum + Stigonema, Hapalosiphon, Scytonema, & Nodularia Sphagnum girgensohnii + Anabaenopsis

Rate
0.42 nmol N cm-2 hr-1 1.87 nmol N gdm-1 hr-1 8.2 nmol N gdm-1 hr-1 1.23 nmol N gdm hr
-1 -1

Reference
Rodgers & Henriksson 1976 Brasell et al. 1986 Brasell et al. 1986 Brasell et al. 1986 Brasell et al. 1986 Brasell et al. 1986 Brasell et al. 1986 Brasell et al. 1986 Vlassak et al. 1973 Morimoto & Maruyama 1982 Morimoto & Maruyama 1982 Basilier 1979 Basilier 1979 Basilier 1979 Basilier 1979 Basilier 1979 Basilier 1979 Snyder & Wullstein 1973b Snyder & Wullstein 1973b Englund 1976 Englund 1976 Lambert & Reiners 1979 Granhall & Selander 1973 Lambert & Reiners 1979 Lambert & Reiners 1979 Lambert & Reiners 1979 Lambert & Reiners 1979 Lambert & Reiners 1979 Lambert & Reiners 1979 Lambert & Reiners 1979 Lambert & Reiners 1979

3.1 nmol N gdm-1 hr-1 3.27 nmol N gdm-1 hr-1 2.57 nmol N gdm-1 hr-1 3.27 nmol N gdm-1 hr-1 10.4 nmol N gdm hr
-1 -1

0.13 nmol N gfm-1 hr-1 0.13 nmol N gfm-1 hr-1

None detected *included only plant apex. 0.033 nmol N gdm-1 hr-1 Sphagnum papillosum + endophytic Nostoc (only plant apex) 43.3 nmol N gdm-1 hr-1 Sphagnum angustifolium + endophytic Nostoc (only plant apex) Drepanocladus aduncus + unidentified epiphytic 25.67 nmol N gdm-1 Cyanobacteria hr-1 (only plant apex) 26.67 nmol N gdm-1 Sphagnum riparium + epiphytic Hapalosiphon hr-1 (only plant apex) 15.3 nmol N gdm-1 hr-1 Sphagnum annulatum + Nostoc (only plant apex) Grimmia + Azotobacter Syntrichia ruralis + Azotobacter Grimmia + Anabaena & Nostoc Racomitrium + Anabaena & Nostoc Sphagnum + Cyanobacteria Sphagnum lindbergii + Nostoc & Scytonema Sphagnum + Cyanobacteria Sphagnum + Cyanobacteria Atrichum + Cyanobacteria Dicranum + Cyanobacteria Pleurozium schreberi + Cyanobacteria Plagiomnium cuspidatum + Cyanobacteria Polytrichum + Cyanobacteria Bazzania trilobata + Cyanobacteria 0.065 nmol N gdm-1 hr-1 0.061 nmol N gdm-1 hr-1 0.13 nmol N/20 cm plant hr-1 0.1 nmol N/20 cm plant hr-1 0.743 nmol N gdm-1 hr-1 1.3 nmol N gdm-1 hr-1 0.29 nmol N gdm-1 hr-1 0.13 nmol N gdm hr
-1 -1

0.053 nmol N gdm-1 hr-1 0.023 nmol N gdm-1 hr-1 0.026 nmol N gdm hr
-1 -1

0.15 nmol N gdm-1 hr-1 0.011 nmol N gdm-1 hr-1 0.033 nmol N gdm-1 hr-1

24 Subalpine Subalpine Alpine zone of Mt. Fuji Antarctic Antarctic Antarctic Antarctic Antarctic Coniferous forest floor Coniferous forest floor Mountain summit East Ongul Island, Antarctica. Sand near a rocky peak. Marion Island (highly minerotrophic receiving nutrient-rich mire runoff)

Chapter 8-3: Nutrient Relations: Nitrogen Feather mosses Sphagnum Aongstroemia fuji-alpina, Ceratodon purpureus, & Bryum + Nostoc Ceratodon purpureus & Bryum pseudotriquetrum + Nostoc 0.23 nmol N gdm-1 hr-1 7.47 nmol N gdm-1 hr-1 3.4 nmol N cm-2 hr-1 2.37 nmol N cm-2 hr-1 Granhall & Lindberg 1978 Granhall & Lindberg 1978 Nakatsubo & Ohtani 1991 Nakatsubo & Ino 1987 Smith & Russell 1982 Smith & Russell 1982 Smith & Russell 1982 Broady et al. 1987

Brachythecium subplicatum + Anabaena, Calothrix, Hapalosiphon, Nostoc, Sphaerocystis, 103.5 nmol N gdm-1 hr-1 Stigonema, & Tolypothrix Ditrichum strictum (balls) + Anabaena, Marion Island (exposed Calothrix, Hapalosiphon, Nostoc, Sphaerocystis, 0.12 nmol N gdm-1 hr-1 wind-swept rocky ridges) Stigonema, & Tolypothrix Grimmia falcate + Anabaena, Calothrix, Marion Island (submerged) Hapalosiphon, Nostoc, Sphaerocystis, Stigonema, 5.15 nmol N gdm-1 hr-1 & Tolypothrix Fumaroles near summit of Campylopus pyriformis & Cephaloziella 11 nmol N gdm-1 d-1 Mt. Melbourne exiliflora + Mastigocladus laminosus

Peatland Associations Sphagnum is highly colonized by a variety of Cyanobacteria, both on its surface (Hooper 1982), and in its hyaline cells (Figure 16; Granhall & Hofsten 1976; Granhall & Lindberg 1978), especially by Nostoc and Hapalosiphon (Sheridan 1975). In bogs and fens, Cyanobacteria on bryophyte surfaces can contribute considerable usable N to the ecosystem (Alexander et al. 1974; Basilier et al. 1978, Basilier 1979; Lambert & Reiners 1979; Rosswall & Granhall 1980, Hooper 1982). Chapman and Hemond (1982) determined that the contribution was greater than that from the only other known input, bulk precipitation (as NO3-). Three types of Sphagnum associations fix N: epiphytic Cyanobacteria, intracellular Cyanobacteria, and N-fixing bacteria (Granhall & Selander 1973, Granhall & Hofsten 1976). Basilier (1979) reported N-fixation activity by Cyanobacteria on Sphagnum, Drepanocladus, and Calliergon in phosphorus-rich environments. Basilier and coworkers (1978), as well as Granhall and Selander (1973), found that the highest N fixation rates in their studies occurred on the mosses Sphagnum and Drepanocladus (s.l.), with a mean value of 9.4 g m-2 yr-1. In fact, Cyanobacteria associated with Sphagnum can have higher N fixation per heterocyst than do free-living Cyanobacteria in the same condition (Basilier 1980). Granhall and Lindberg (1978) reported a total rate of 0.8-3.8 g fixed N m-2 yr-1 in wet Sphagnum communities in a mixed pine and spruce forest in central Sweden. Zimicki (1976) and Basilier et al. (1978) have estimated N fixation in various sites for Sphagnum riparium to be 0.5-6.4 g m-2 yr-1. Basilier et al. (1978) found that the fixation rate in the Sphagnum riparium association was strongly light dependent, but that pH in the range of 4.3 to 6.8 had little effect. Maximum fixation occurred around noon with the middle of the growing season exhibiting the highest rates. Interestingly, they found that rates on the apical portions and non-green portions of the Sphagnum were lower, and that the highest rates occurred on the periphery of the moss community. On the other hand, using 15N as a tracer, Basilier (1980) later found that enrichment of N from Cyanobacteria fixation appeared within two hours in the apex of Sphagnum. It appears that habitat comparisons need to be made to determine where the highest rates might occur.

Figure 16. Potential interactions of micro-organisms within the hyaline cell of Sphagnum. Redrawn from Granhall & Hofsten (1976).

Once the Cyanobacteria convert the N to NH4+ and amino acids, these are available not only for the bryophytes they occupy, but also for the tracheophytes rooted among them. In Thoreau's Bog in Massachusetts, N fixation exceeded atmospheric N deposition (Hemond 1983), and Hemond concluded that microbial N fixation provides sufficient quantity of N that N may never be limiting to primary productivity in a bog (or poor fen) ecosystem. Liverwort Symbiosis Several attempts have been made to explain the high degree of N fixation in liverwort associations. In an early attempt, Griggs (1937) grew liverworts from Katmai volcanic ash on N-free sand for three years to determine their success compared to that of liverworts on the same medium, but with the addition of 4 mg/L NH4NO3. During that three-year period, the ones with the additive grew no better, but toward the end of the three years, the N-free cultures became pale and unhealthy. When 4 mg/L NH4NO3 was added to the N-free cultures, they promptly revived. Griggs took this as evidence that no N fixation had occurred. Nevertheless, at least the thallose liverwort Blasia pusilla has symbiotic Cyanobacteria that do perform N

Chapter 8-3: Nutrient Relations: Nitrogen

25

fixation (Rodgers 1978; Peters 1991; Figure 17). In fact, there are many genetic strains of Nostoc associated with Blasia (West & Adams 1997; Costa et al. 2001). The presence of Nostoc induces both structural and metabolic changes within the Blasia thallus (Kimura & Nakano 1990; Meeks 1990).

low in usable N. The ability to colonize rapidly, symbiont intact, is facilitated in Blasia pusilla by the production of two types of gemmae. These gemmae permit the symbiont to travel with the gemma and easily renew the partnership arrangement upon germination (Renzaglia 1982b; Duckett & Renzaglia 1993). Taxa that depend on spores for their dispersal would not benefit from this convenience. Hornwort Associations Hornworts (Anthocerotophyta) are well known for their symbiotic associations with Cyanobacteria, especially Nostoc in association with Anthoceros and Phaeoceros (Peirce 1906; Ridgway 1967; Enderlin & Meeks 1983; Steinberg & Meeks 1987). A wide diversity of Nostoc strains infect these hornworts (West & Adams 1997), and, it appears that Anthoceros harbors a Nostoc that is unique from that of Blasia (Leizerovich et al. 1990). But Phaeoceros (Figure 18) also hosts the filamentous Calothrix (Cyanobacteria) (West & Adams 1997); this multiplicity of symbiotic genera is apparently unusual; Rai et al. (2000) indicate that typically only one genus will infect a particular taxonomic group of plants.

Figure 17. Blasia pusilla. Arrow indicates Nostoc colony. Photo by Paul Davison, University of North Alabama.

Nostoc is only capable of invading the liverwort when the Nostoc is in its mobile stage (Kimura & Nakano 1990). That is, when the segments (called hormogonia) of a filament separate, they are mobile by a gelatinous sol-gel transformation that permits them to slither and glide. In this stage they are able to invade the thallus of B. pusilla and induce the morphological changes that permit the partnership to work. At the same time, the B. pusilla signals the Nostoc by producing two auricles (earlike lobes), each with an enclosed chamber housing a slime papilla that fills the chamber with mucilage (Renzaglia 1982a). The mucilage attracts the Nostoc, which then takes up residence in the chamber. Once the Nostoc arrives, the auricle increases in size and closes its opening. Following the invasion, the surrounding cells of the Blasia thallus have attenuated growth and produce branched filaments from hyaline cells that penetrate the Nostoc colonies (Kimura & Nakano 1990). These filaments form a labyrinth of wall ingrowths into the Nostoc cells, suggesting that they may have the role of transfer cells for exchanging metabolites (Ridgway 1967; Duckett et al. 1977). Once it has settled into its thallus home, the Nostoc produces numerous heterocysts, which are essential for the N fixation. When the Nostoc grows deeply embedded within the liverwort thallus, it no longer has access to dissolved CO2. Stewart and Rodgers (1977; 1978) determined that the Nostoc obtains its carbon through transfer from the Blasia thallus to Nostoc, suggesting that this is really a mutualistic relationship. Within the thallus the Nostoc requires a higher light intensity and higher temperature (above 17C) for maximal activity than when living alone (max activity above 12C) (Rodgers 1978). Hence, the liverwort provides a safe compartment that will remain moist much longer than the external environment, and even provides the needed carbon source for its symbiont. The presence of these Nostoc symbionts in liverworts seems to be restricted to taxa that are pioneers (Schuster 1992a, b)., living in temporary habitats that are likely to be

Figure 18. Phaeoceros carolinianus showing bluish green color typical of plants with Nostoc inhabitants. Photo by Michael Lth.

For the association to begin, the Nostoc must form hormogonia that can break away and move through the environment to reach the hornwort (Wong & Meeks 2002), just as in Blasia. But it seems that the hornwort makes certain that this occurs, if there is Nostoc in the vicinity. Free-living Nostoc rapidly forms hormogonia when in the presence of Anthoceros punctatus, or even in the presence of agar preconditioned with A. punctatus (Campbell & Meeks 1989), indicating a diffusable substance from A. punctatus that stimulates this response. Both Nostoc and the hornwort seem to be modified physiologically once joining in symbiosis (Joseph & Meeks 1987; Campbell & Meeks 1992). Before the partnership can work, the Nostoc must form heterocysts (Wong & Meeks 2002). This is where the enzyme nitrogenase, needed for the N fixation, is located in both free-living and symbiotic strains (Rai et al. 1989). When mutants of Nostoc punctiforme, unable to form heterocysts, were introduced to Anthoceros punctatus, the partnership formed, but no N fixation occurred; the mutants did not produce any nitrogenase. As in the Blasia symbionts, the nitrogenase of the Nostoc must have an anaerobic environment in which to fix

26

Chapter 8-3: Nutrient Relations: Nitrogen

nitrogen. Campbell and Meeks (1992) demonstrated this by showing that the symbiont could produce fixed N only under anaerobic conditions when grown outside its host. However, when it grew in its Anthoceros punctatus host, it could be grown aerobically; the special cavities where it grew on the host provided the anaerobic conditions needed. Perhaps one explanation for the success of N fixation within the host lies in the structure of the symbiont heterocyst, contrasting with that of the free-living Nostoc strains. When growing inside the host, the Nostoc heterocyst lacks the outer polysaccharide layer typical of free-living Nostoc (Campbell & Meeks 1992). Rather, it appears that when the Nostoc grows in the cavities of Anthoceros punctatus, the cavities replace that wall function. Anthoceros also mediates the nitrogenase activity, supressing it in the presence of NO3- (Campbell & Meeks 1992) and NH4+ (Steinberg & Meeks 1991). The end product of the Nostoc fixation is NH4+, accounting for 75% of the introduced radioactive N after 0.5 min, but only 14% after 10 minutes of incubation (Meeks et al. 1985), indicating a rapid transformation to something else. Glutamine and glutamate are quickly synthesized via the glutamine synthetase-glutamate synthase pathway, preventing the toxic buildup of NH4+. Thus one end result of the symbiosis is that the intracellular levels of NH4+ are low compared to those of symbiont-free Anthoceros. Only 10% of the NH4+ is assimilated into the Nostoc; 1% is lost to the medium; Anthoceros incorporates the remainder. Prakasham and Rai (1991) demonstrated that there is a specific methylammonium transport system in the symbiotic Nostoc, which may account for the reduced NH4+ levels and rapid transfer to the host. In symbiont-free Anthoceros supplied with high levels of NH4+, the glutamate dehydrogenase system is functional, permitting an NH4+ buildup (Meeks et al. 1983). Therefore, it appears that the Nostoc partner provides a very effective and safe source of NH4+ for the Anthoceros host (Meeks et al. 1985). As in the Blasia partnership, Nostoc living within the hornwort gets its carbon primarily from its host plant (Stewart & Rodgers 1977). In fact, Nostoc isolated from Anthoceros punctatus had only 12% of the Rubisco activity of free-living strains, with an equal reduction in CO2 fixation (Steinberg & Meeks 1989; Rai et al. 1989). However, the distribution and levels of Rubisco were similar in the two strains (Rai et al. 1989), with 4.3% and 5.2% of the protein as Rubisco in symbionts and free-living Nostoc, respectively (Steinberg & Meeks 1989), suggesting that there is regulation of the Rubisco activity and not an alteration at the gene transcription level. This could be related to the fact that the structure of the chloroplast differs somewhat; the Nostoc contains the typical cyanophycean granules, but it lacks phycobilisomes, the cellular organelle located on the surface of the thylakoids of the chloroplasts and in which the biliprotein pigments (phycocyanin, phycoerythrin) are present (Honegger 1980). Because the Nostoc has reduced ability to fix its own carbon, this transfer of fixed carbon from A. punctatus to Nostoc is necessary for the fixation of N2. When the Nostoc-hornwort association was deprived of light for 28 hours, the rate of acetylene reduction (as a measure of N fixation) declined by 99%, but resumed up to 64% of its

illuminated activity when supplied with glucose in the dark (Steinberg & Meeks 1991), indicating the need for light and photosynthetic activity for the partnership to work. When gametophytes were deprived of light, but sporophytes were provided with light, nitrogenase activity continued (Stewart & Rodgers 1977), suggesting a transfer of sugar from the sporophyte to the gametophyte, then to the Nostoc. These factors suggest that the Nostoc, living in the reduced light of the interior of the hornwort thallus, may be dependent upon the hornwort for glucose or similar carbohydrate as an energy source in order to continue its N fixation, thus completing a true mutualistic relationship with its host. The local sites of the host plants act as islands that effectively keep the Nostoc strains in isolation. Even within a single host plant there may be a great diversity of cyanobacterial strains, and these strains seem to be restricted to one site (Costa et al. 2001). Nevertheless, some host plants shared strains of Nostoc that could be found growing 2000 m away. Furthermore, strains found in Blasia could also be found in the lichen Peltigera neopolydactyla. Although different cavities can easily host different strains in both Blasia and the Anthocerotophyta, a single cavity seems only to host one strain. Lunar Rocks Liverworts were among the few organisms to grow successfully on lunar rocks. But why? Marchantia polymorpha exhibited a tremendous increase in growth following being sprinkled with Apollo 11 or 12 lunar rock material. Hoffman (1974) followed up on this observation by testing the effects of basalt from Minnesota and Chorizon substrate from the Valley of Ten Thousand Smokes, Alaska. In both cases, the growth of M. polymorpha was significantly increased. But what caused this surge of growth? Nitrogen was absent in any form in both the lunar material and the basalt, and neither P nor K was abundant, so the three typical fertilizer nutrients seem not to be the cause. The macronutrients Ca, Mg, and S were all more abundant in basalt than in the C-horizon soil, but the C-horizon soil caused the greater stimulation. Iron remains a possibility, being abundant in all three substrata. We already know that it stimulates the growth of Funaria hygrometrica (Hoffman 1966). On the other hand, no data were gathered on the pH, which could affect the solubility, and therefore availability, of all the nutrients. Some have speculated that survival of the liverwort was possible due to partnering Cyanobacteria that could trap and convert the atmospheric nitrogen. Perhaps we need to look for soil and rock components that foster the N fixation reaction.

N Enrichment
Many studies in peatlands have included enrichment of N to determine effects on bryophyte productivity. In the high Arctic heath, Gordon et al. (2001) found that applications of N (0, 10 & 50 kg ha-1 yr-1) and P (0 & 5 kg ha-1 yr-1) caused a decrease in lichen cover; applications of 10 kg ha-1 yr-1 resulted in a higher proportion of physiologically active bryophyte shoots. Nevertheless, individual bryophyte species responded differently, suggesting that we cannot draw generalizations from limited fertilization experiments.

Chapter 8-3: Nutrient Relations: Nitrogen

27

Summary
Nitrogen is available to bryophytes as ammonium (NH4+), nitrite (NO2-), nitrate (NO3-), and organic forms such as amino acids and urea. Nitrite, however, is generally toxic. Ammonium can lower internal pH and suppress growth. Nitrite can cause an increase in chlorophyll a, whereas nitrate can cause a decrease in chlorophyll b, both causing an increase in the a/b ratio. But effects on amino acid and protein concentration vary among species and among habitats. In the Arctic, amino acids and urea are utilized by both bryophytes and tracheophytes. Sphagnum often seems to benefit more from amino acids than from ammonium. N deficiency, or the wrong form of N (e.g. NH4+), can cause bryophytes to become long and thin, appearing etiolated. Glycine, serine, arginine, and alanine can induce branching. Methionine not only did not induce branching, but it also inhibited growth. Glycine caused the greatest weight and length gain of these amino acids in Java moss. Even nucleic acids are usable N sources, with good leafy shoot growth in adenine and guanine, but no growth in uracil or thymine in some species and good growth in others. In Sphagnum squarrosum uric acid and cytosine caused the plant to be come thalloid. Some, perhaps many, bryophytes solve the nitrogen problem through symbiotic partners, especially Cyanobacteria, that carry out nitrogen fixation. This process seems to be especially important in the polar and alpine regions under warmer summer conditions up to ~25C. But more xeric conditions such as among epiphyllous tropical bryophytes and associated with prairie and grassland cryptogamic crusts also benefit from N fixation. In all of these habitats, bryophytes have an important role in maintaining the moisture necessary for the fixation to occur. Peatlands have a high N fixation rate, and Cyanobacteria are common in association with Sphagnum. They have a wider pH tolerance range (4.36.8) than the Cyanobacteria in the cold habitats (5.96.2). The liverwort Blasia pusilla provides a special chamber in each auricle where it is moist with mucilage and the Cyanobacteria enter and grow. It then seals the chamber and produces filaments that penetrate the Nostoc colonies. Finally the Nostoc produces numerous heterocysts. The Nostoc even travels with the gemmae. Anthoceros punctatus forms a similar partnership, as do most of the hornworts, but it even stimulates the Nostoc to form hormogonia, permitting it to slither toward the hornwort. In both liverwort and hornwort partnerships, the ammonium produced by the Cyanobacterial heterocyst is quickly converted to glutamine and glutamate to avoid the buildup of toxic ammonium. The Anthoceros gets almost 90% of the fixed N and provides fixed C to its Cyanobacteria partner. Moon rock, and rock taken from volcanic areas on Earth, stimulate the growth of bryophytes, but we don't know why. One possibility is the high concentration of iron; another is that symbionts thrived on these rocks, providing N fixation.

It appears that bryophytes play a major role as a substrate for N fixation in many nutrient-poor habitats, making than essential component of those ecosystems.

Acknowledgments
I appreciate the contributions of undergraduate Phil Gaudette and M. S. student Jennifer Jermalowicz Jones for their critical reading of the manuscript from the perspectives of students interested in nutrient relationships of bryophytes. Medora Burke-Scoll prepared the table on N contributions by Cyanobacteria.

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